Structural insights into the AFB1 aptamer coupled with a rationally designed CRISPR/Cas12a-Exo III aptasensor for AFB1 detection

Abstract

Aflatoxin B₁ (AFB₁) is a typical food contaminant. A truncated DNA aptamer of AFB₁ was reported by our team in previous work. However, the recognition mechanism between aptamer and AFB₁ was lacking, which was crucial for the design of related aptasensor. Herein, the binding of aptamer to AFB₁ was systematically studied and found that it was an exothermic process and the conformation of aptamer changed during the recognition process. Loop bases in the secondary structure of aptamer formed a special binding pocket to recognize AFB₁. Van der Waals and electrostatic interaction were the main driving forces. By blocking the stem bases guided by the structural investigation, a rationally designed CRISPR/Cas12a-Exo III aptasensor for AFB₁ detection was constructed, and the sensitivity was improved by target recycling. Under optimal conditions, the linear detection range for AFB₁ was 0.01-20 ng/mL, and AFB₁ was accurately determined in corn and wheat samples. This work laid a theoretical foundation for the design of AFB₁ aptasensor, and the developed detection model came up with new ideas for the development of CRISPR/Cas12a-based aptasensor.

Keywords: AFB(1) aptamer; Binding mechanism; CRISPR/Cas12a-Exo III.